The present invention is directed to a process for culturing cells in a bioreactor having semi-permeable membranes and more specifically, to a process involving the provision of circulation means on the cell side of the membranes to improve mixing and reduce boundary layers and concentration gradients along the membranes and in and/or around the cell mass to improve mass transfer.
Cell culture devices for culturing cells in vitro having a shell with a plurality of hollow fiber membranes have been known for quite some time. Media containing oxygen, nutrients and other chemical stimuli is transported through the lumen of the hollow fiber membranes and undergoes a pressure drop resulting in an outward radial convective flow at the entry port of the device and an inward flow at the exit port of the device. Cells are grown in the fluid space between the fibers and the shell wall. Cell culture devices using sheet membrane bioreaactors are also well known and work as well as hollow fiber membranes.
Hollow fiber culture devices have been proven to be ideal for the maintenance of many types of cells at high densities in vitro. The mass transfer characteristics of hollow fiber culture devices provide an efficient means for delivering nutrients and removing waste products from a culture. The semi-porous, hollow fiber membranes can be selected with various pore sizes. With proper pore size selection, the cellular product can be maintained on the outside of the fibers while waste products and contaminating proteins will pass through the membrane pores into the lumen of the hollow fibers where they can be subsequently removed from the culture. In the use of such conventional hollow fiber techniques, the extracapillary space is capped or dead-ended at both ends. This creates a Starling effect and a heterogeneous environment. Cells generally grow only toward the proximal end of the bioreactor. Also, large molecular weight components trapped in the extracapillary space concentrate at the distal end.
To economically produce cell-derived products in a hollow fiber device, large numbers of the cells must be maintained viable in optimal culture conditions for product formation over long periods of time. Prior art hollow fiber culture devices have many limitations that prevent their use in the economical production of cell-derived products in commercial quantities. These limitations include:
1) formation of gradients in the cell compartment; PA0 2) inability to directly monitor and control cellular environment; PA0 3) lack of fluid movement in cell compartment leads to microenvironment formation around cells; PA0 4) fibers are not equidistant apart in culture device leading to anoxic or dead spaces; PA0 5) efficient mass transfer becomes difficult at high cell densities; and PA0 6) the pressure drop across the device increases as the pressure is scaled up, increasing the problems cited above, thus limiting scaleability.
The purpose of the present invention is to overcome these limitations, making it possible to utilize a hollow fiber culture device or other conventional sheet membrane devices for the economical production of cell-derived products
In order to overcome the foregoing limitations, it was proposed in the Cracauer et al. U.S. Pat. No. 4,804,628 to provide an expansion chamber connected to opposite ends of the extracapillary space by cell culturing units such as a hollow fiber cartridge in which the cells are maintained and grown. The hollow fiber cartridge included a plurality of capillaries extending through a shell with each capillary including a lumen through which media containing oxygen, nutrients and other chemical components are transported. A delivery system or integration circuit delivers a primary media supply through the lumens of the capillaries of the bioreactor as shown in the prior art schematic diagram in FIG. 2. The integration circuit includes a pump for circulating the media through the integration circuit. A gas exchange cartridge, a DO probe, a pH probe, and an integration chamber may also be included in the integration circuit.
The extracapillary circuit containing the expansion chamber may be provided with valves between the expansion chamber and the extracapillary space. By regulating pressure differences between the integration circuit chamber and the expansion circuit chamber, a cyclic flow of media is generated across the hollow fiber membranes and through the extracapillary space of the bioreactor shown in FIG. 2. As the integration circuit chamber is pressurized above the expansion circuit chamber, media is pushed from the lumens of the fibers across the fiber membranes into the extracapillary space along the full length of the bioreactor. This bathes the cells in nutrient and oxygen rich media. As the expansion chamber is pressurized above the integration chamber, oxygen depleted media and waste by-products are moved from the expansion circuit, through the cell bed and finer membrane into the integration circuit. This process in continually repeated during the cell culture period and results in a substantially homogeneous environment. Unfortunately, as the media moves across the fiber from the integration chamber to the expansion chamber, cells are pushed out from the bioreactor into the expansion chamber. There the cells will settle out, become trapped and die.